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M T Santamarina

Publications and source records attributed to M T Santamarina.

At least 19 recordsLinked to original sources
PubMed2006

Scuticociliate proteinases may modulate turbot immune response by inducing apoptosis in pronephric leucocytes.

The role of proteinases of the histiophagous ciliate Philasterides dicentrarchi, purified by affinity chromatography in bacitracin-Sepharose, on apoptosis (programmed cell death) of turbot pronephric leucocytes (PL) was investigated. The results showed that more than 90% of proteinases purified by bacitracin-Sepharose were cysteine proteinases, which lacked significant caspase-3-like activity and generated three main gelatinolytic bands of molecular weights 36, 45 and 77 kDa as determined by gelatine-SDS-PAGE and immunoblot. Viability of PL cells after 24 h stimulation with P. dicentrarchi cysteine proteinases did not differ from that of non-stimulated cells. Apoptosis was confirmed by: (i) caspase activity, (ii) DNA fragmentation, and (iii) nucleus fragmentation. The caspase-3-like activity in PL incubated for 4h in the presence of 125, 250 and 500 microg/ml of proteinases increased in a dose-dependent fashion. The PL DNA was fragmented following 24-h exposure to P. dicentrarchi cysteine proteinases and characteristic DNA ladders consisting of multimers of approximately 180-200 pb were produced. Morphological changes, such as chromatin condensation and nucleus fragmentation, were observed under fluorescence microscopy after DAPI staining of the PL cells incubated with cysteine proteinase-incubated for 24 h. The results suggest that the pathogenic scuticociliate P. dicentrarchi may induce host leucocyte programmed cell death via the production of cysteine proteinases, as a mechanism of pathogenesis and evasion of the turbot innate immune response.

Animals↗
PubMed2001

Unusual electrophoretic patterns for phosphoglucomutase and fumarase in a population of Lecithochirium rufoviride (Trematoda: Hemiuridae), a parasite of Conger conger.

Electrophoretic analyses of phosphoglucomutase (PGM) and fumarase (FH) in a population of Lecithochirium rufoviride parasitizing Conger conger, revealed 2 independent activity zones for each enzyme on starch gel electrophoresis. However, some individuals exhibited only 1 activity zone for 1 or both enzymes. The banding patterns observed strongly suggest that (1) PGM is coded by 2 polymorphic loci, Pgm-1 (expressed in all individuals) with allelic frequencies not significantly different from those expected under Hardy-Weinberg equilibrium, and Pgm-2 (expressed in a subset of individuals); and (2) FH is coded by 2 loci, Fh-2 (monomorphic and expressed in all individuals) and Fh-1 (expressed in a subset of individuals). A high degree of concordance (88.75%) was observed between the expression and nonexpression of Pgm-2 and Fh-1. The most likely explanations for these findings are either variation in enzyme expression with developmental stage or the presence of null alleles at high frequencies in the population.

Animals↗
PubMed1999

Characterization by enzyme electrophoresis of specimens of the genus Helicometra (Trematoda, Opecoelidae) from fish caught off the coast of northwest Spain.

Species within the genus Helicometra are difficult to distinguish on morphological grounds alone, and are best discriminated with the aid of biochemical techniques. In the work reported here, the electrophoretic mobility of malate dehydrogenase isoenzyme of the Mdh-1 locus was used to characterize 375 individuals of Helicometra obtained from various teleost species (Anguilla anguilla, Conger conger, Gobius niger or Ciliata mustela) caught off the coast of northwest Spain. The results suggest that all specimens belong to only one species, probably H. fasciata. Observed genotype frequencies did not differ from those expected under the assumptions of Hardy-Weinberg equilibrium, even when the genic frequencies differ considerably respecting a Mediterranean population.

Alleles↗
PubMed1998

Oral pharmacological treatments for parasitic diseases of rainbow trout Oncorhynchus mykiss. II. Gyrodactylus sp.

A total of 24 drugs were evaluated as regards their efficacy for oral treatment of gyrodactylosis in rainbow trout Oncorhynchus mykiss. In preliminary trials, all drugs were supplied to infected fish at 40 g per kg of feed for 10 d. Twenty-two of the drugs tested (aminosidine, amprolium, benznidazole, bithionol, chloroquine, diethylcarbamazine, flubendazole, levamisole, mebendazole, metronidazole, niclosamide, nitroxynil, oxibendazole, parbendazole, piperazine, praziquantel, ronidazole, secnidazole, tetramisole, thiophanate, toltrazuril and trichlorfon) were ineffective. Triclabendazole and nitroscanate completely eliminated the infection. Triclabendazole was effective only at the screening dosage (40 g per kg of feed for 10 d), while nitroscanate was effective at dosages as low as 0.6 g per kg of feed for 1 d.

Administration, Oral↗
PubMed1998

Oral pharmacological treatments for parasitic diseases of rainbow trout oncorhynchus mykiss. III. Ichthyobodo necator.

A total of 32 drugs were evaluated as regards their efficacy for oral treatment of Ichthyobodo necator infestation of rainbow trout. In preliminary trials, all drugs were supplied to infected fish at 40 g per kg of feed for 10 d. The majority of the drugs tested (1,3-di-6-quinolylurea, aminosidine, amprolium, benznidazole, bithionol, chloroquine, diethylcarbamazine, dimetridazole, diminazene aceturate, febantel, flubendazole, ketoconazole, levamisole, mebendazole, netobimin, niclosamide, niridazole, nitroscanate, nitroxynil, oxibendazole, parbendazole, piperazine, praziquantel, ronidazole, sulphaquinoxaline, tetramisole, thiophanate, toltrazuril and trichlorfon) were ineffectdive. Metronidazole and secnidazole were 100% effective (unlike the other nitroimidazoles tested, namely dimetridazole, benznidazole and ronidazole). The non-carbamate benzimidazole triclabendazole was likewise 100% effective.

Administration, Oral↗
PubMed1998

Oral pharmacological treatments for parasitic diseases of rainbow trout Oncorhynchus mykiss. I: Hexamita salmonis.

Various drugs were evaluated as regards efficacy for the treatment of Hexamita salmonis infection in rainbow trout. The results confirm the efficacy of nitroimidazoles: infection was completely eradicated not only by metronidazole (which has been recommended previously for the treatment of hexamitosis), but also by benznidazole, ronidazole and secnidazole, which have not been assayed previously. The non-nitroimidazoles albendazole, aminosidine, diethylcarbamazine and nitroscanate also completely eliminated infection. The remaining non-nitroimidazoles tested (amprolium, bithionol, febantel, flubendazole, levamisole, netobimin, niclosamide, nitroxynil, oxibendazole, parbendazole, piperazine, praziquentel, tetramisole, thiophanate, toltrazuril, trichlorfon and triclabendazole) were not effective.

Administration, Oral↗
PubMed1997

Monoclonal antibodies against diagnostic Anisakis simplex antigens.

Five monoclonal antibodies (UA2, UA3, UA5, UA6, and UA8) specific for Anisakis simplex are described. All are IgG1/kappa monoclonal antibodies, except for UA2, which is an antibody IgM/kappa. The molecular weights of the major components recognized in immunoblotting are 48 and 67 kDa (UA2); 139 kDa (UA3 and UA5; same epitope); 35, 38, and 139 kDa (UA6); and 205 kDa (UA8). UA2 was the only monoclonal antibody to recognize both components of an excretion-secretion antigen preparation and antigens in the excretory cell and esophageal glands of third-stage A. simplex larvae; antigens in the excretory cell were also recognized by UA3 and UA6. Cross-reactivity studies using a hyperimmune polyclonal rabbit serum reacting with various ascaridoid nematodes indicated that the antigens captured by our monoclonal antibodies were specific for A. simplex. Finally, comparative studies of our monoclonal antibodies and An2 (the only monoclonal antibody currently available for serodiagnosis of human anisakiasis), based on the calculation of multiples of normal activity for human anisakiasis sera, indicated that our monoclonal antibodies (and particularly UA3) recognized antigens that are good candidates for serodiagnostic purposes.

Animals↗
PubMed1996

Antigenic cross-reactivity in mice between third-stage larvae of Anisakis simplex and other nematodes.

We used ELISA and immunoblotting to investigate antigenic cross-reactivity in mice between third-stage larvae of Anisakis simplex and five other nematodes: the ascaridoids Ascaris suum, Toxocara canis and Hysterothylacium aduncum, and the nonascaridoids Trichinella spiralis and Trichuris muris. Two sera were raised against each species (including A. simplex, but excluding A. suum), by infection or by immunization with somatic antigens. Serum against A. suum was raised by immunization only. The reactivities of each serum with A. simplex somatic antigens (SA), excretion-secretion antigens (ES), pseudocoelomic fluid antigens (PF) and cuticular antigens (CA) were investigated. The results of ELISA indicated high antigenic cross-reactivity between A. simplex and the remaining ascaridoid nematodes, confirming that there is extensive antigenic similarity within this group of nematode parasites. Immunoblotting again confirmed the high degree of cross-reactivity between the SA of A. simplex and SAs of the other ascaridoids, although several A. simplex SA components in the 11-18 kDA range were only recognized by sera from mice infected with A. simplex. In addition, two A. simplex PF components of 22 and 27 kDA, were recognized only by sera from mice infected with, or immunized with the SA of, A. simplex. Finally, the anti-phosphorylcholine monoclonal antibody BH8 recognized only a small number of A. simplex antigens, indicating that phosphorylcholine epitopes are not significant contributors to the observed cross-reactivity with the other nematodes.

Animals↗
PubMed1996

Free and bound biotin molecules in helminths: a source of artifacts for avidin biotin-based immunoassays.

The avidin-biotin molecular recognition system is widely used in parasite immunology. However, the presence of biotin and/or biotin-containing molecules (BCMs) in samples may lead to erroneous results. In the work reported herein we investigated the extent to which biotin and BCMs present in helminth extracts may interfere in avidin/biotin-based immunoassays and developed an enzyme-linked immunosorbent assay (ELISA) for quantification of these components. In avidin-based ELISA using antinematode monoclonal antibodies, an extract of the nematode Anisakis simplex showed very high background reactivity due to biotin/BCMs, whereas the background reactivity in an extract of the nematode Trichinella spiralis was negligible. To investigate interspecies differences further, we performed Western-blot analyses (with avidin as the detector) of extracts from seven nematodes (A. simplex, Ascaris suum, Toxocara canis, Hysterothylacium aduncum, T. spiralis, and Trichuris muris) and the cestode Bothriocephalus scorpii. Even within superfamilies there was considerable variation in the banding patterns obtained. The above-mentioned results confirm that biotin and BCMs may be a significant source of interference in ELISA and immunoblotting, two of the techniques most widely used in parasitological immunodiagnosis. A competition ELISA designed to allow accurate quantification of biotin and BCMs in helminth extracts likewise indicated very considerable interspecies variation. Both A. simplex and H. aduncum had very high biotin/BCM contents. Microdialysis of extracts in the presence of dimethylsulfoxide to remove free biotin prior to ELISA indicated that the high biotin/BCM content of the H. aduncum extract (but not the A. simplex extract) was very largely due to free biotin. Taken together, these results indicate that extreme caution should be exercised in the use of avidin/biotin-based immunoassays for the detection of helminth antigens and that in many cases it may be better to use an alternative recognition system.

Animals↗
PubMed1996

The humoral immune response of turbot, Scophthalmus maximus L., to spore-surface antigens of microsporidian parasites.

Experiments based on enzyme-linked immunosorbent assay (ELISA) revealed considerable antigenic homology in turbot between two species of microsporidian, Tetramicra brevifilum (a parasite of the turbot, Scophthalmus maximus) and Glugea caulleryi (a parasite of the lesser sand-eel, Ammodytes tobianus). We next investigated whether G. caulleryi is able to suppress the turbot immune response. Intraperitoneal inoculation of turbot with G. caulleryi spores (whether heat-killed or viable) did not suppress the humoral immune response to injection of G. caulleryi spores plus adjuvant 15 days later; in fact, specific serum antibody levels (as revealed by ELISA) reached maximum levels by about Day 30 post re-exposure. Similar results were obtained with cellular enzyme-linked immunosorbent assay: 15 days after injection with G. caulleryi spores plus adjuvant, specific antibody secretion rate was higher in turbot which had been pre-exposed to G. caulleryi spores than in turbot which had not been pre-exposed.

Animals↗
PubMed1995

A sandwich immunoassay to quantify low levels of turbot (Scophthalmus maximus) immunoglobulins.

We have developed an enzyme-linked immunosorbent assay for the quantification of turbot (Scophthalmus maximus) immunoglobulin (Ig). The capture antibody is a rabbit polyclonal antiserum to turbot Ig, and the detector antibody a monoclonal antibody (UR3) to the turbot Ig heavy chain. Both antibodies bind nearly 100% of turbot Ig. The assay allows detection of turbot Ig in serum at concentrations as low as 0.16 micrograms ml-1 and takes less than 4 h. Precision is satisfactory, with intra-assay coefficients of variation (CVs) ranging from 2.1 to 16.6%, and inter-assay CVs ranging from 5.8 to 24.6%. We used the assay to determine Ig concentrations in the sera of healthy turbot of different weights. Mean serum Ig concentration was 3.35 +/- 0.74 mg ml-1 for fish weighing 15-25 g and 11.14 +/- 1.87 mg ml-1 for fish weighing 1000-2000 g.

Animals↗
PubMed1995

Anisakis simplex: stage-specific antigens recognized by mice.

To investigate antigenic differences between the developmental stages of Anisakis simplex, somatic, excretion-secretion and detergent-solubilized surface antigen preparations of third- and fourth-stage A. simplex larvae, and a somatic antigen preparation of adults, were characterized by SDS-PAGE and immunoblotting using mouse immune serum containing antibodies against the two larval stages. Excretion-secretion and surface antigen preparations from third-stage larvae behaved very differently from those of fourth-stage larvae, in both SDS-PAGE and immunoblotting; this suggests that certain excretion-secretion and surface antigens are highly stage-specific. By contrast, somatic components of third- and fourth-stage larvae were found to have very similar banding patterns, suggesting conservation of these components during the development of the parasite in mice. The SDS-PAGE and immunoblotting results for adult somatic components seem to support this hypothesis.

Animals↗
PubMed1994

Monoclonal antibodies to turbot (Scophthalmus maximus) immunoglobulins: characterization and applicability in immunoassays.

Five monoclonal antibodies (mAbs) to immunoglobulins (Igs) of the turbot Scophthalmus maximus were produced and characterized. All the mAbs (denominated UR1, UR3, UR4, UR6 and UR7) are of isotype IgG1/kappa and show good anti-turbot Ig reactivity in enzyme-linked immunosorbent assay (ELISA) and immunoblotting. Results of competitive ELISA and immunoblotting analysis indicate that these five mAbs react with at least three different epitopes on the turbot Ig H chain. Except in the case of UR1, reactivity with periodate-treated purified turbot Ig was much lower than with the untreated Ig, suggesting that carbohydrate residues are involved in epitope recognition. All the mAbs showed reactivity with sera from the closely related species Scophthalmus rhombus but not with sera from species of other flatfish genera. One of these mAbs (UR3) has been successfully applied for the detection of antibodies against Vibrio anguillarum in ELISA.

Animals↗
PubMed1993

Requirements for the induction of cross-reactive anti-Trichinella IgE antibodies in mice.

Mice primed with Trichinella spiralis or T. pseudospiralis and reinfected with either the homologous or the heterologous species produced high levels of IgE antibodies that cross-reacted with the non-inducing strain in passive cutaneous anaphylaxis assays. Cross-reactive antibodies were not induced by primary infection. Cross-reactivity persisted for more than 6 months following secondary infection or destruction of encysted larvae with mebendazole. Both the prevention of larvi-position by thiabendazole and the interruption of infection using naphthalophos indicated that the presence of the pre-adult stage alone provided sufficient priming for the induction of detectable levels of cross-reactive IgE by subsequent reinfection. These results suggest the existence of two sets of Trichinella allergens, one comprising species-specific major allergens (MAs) and the other comprising minor allergens (mAs) evoking a cross-reactive IgE response that occurs to a detectable extent only when the response to MAs has reached its ceiling. These findings are relevant to the design of experiments investigating the role played by IgE antibodies in protection against reinfection in rodents.

Animals↗
PubMed1993

Anisakis simplex: antigen recognition and antibody production in experimentally infected mice.

The kinetics of antibody response to intraperitoneal infection of mice with third stage larvae of Anisakis simplex was investigated by ELISA. Maximum antibody response to excretion-secretion (ES) antigens was reached before maximum response to somatic (SA) antigens. Total immunoglobulin (Ig) production (consisting mainly of IgM and IgG1 isotypes) was very similar in both cases. Immunoblotting was used to characterize the antigens recognized by the host in the presence or absence of the metabolic products released by the parasite in vivo. Sera from mice infected with live larvae (anti-L3 L serum) and immunized with dead larvae (anti-L3 D serum) recognized a similar pattern of bands in immunoblots of ES and SA antigen preparations. In the latter, however, three bands at 14, 17 and 18 kD were only recognized by the anti-L3 L serum. A possible explanation is that these low molecular weight antigens are ES products released only in vivo. Finally, the immune response in mouse was compared using ELISA and immunoblotting with the response of a human anisakiasis reference serum, and was found to display considerable similarities. This suggests that the mouse may be a useful model for studying the immunobiology of A. simplex in man.

Animals↗
PubMed1991

Anatomical location of phosphorylcholine and other antigens on encysted Trichinella using immunohistochemistry followed by Wheatley's trichrome stain.

This work investigated the location on the parasite of Trichinella antigens recognized by the mouse immune system and the question as to which of them bear the epitope phosphorylcholine (PC). Wheatley's trichrome stain (initially developed for faecal smears) proved to be excellent for visualization of Trichinella structures, enabling four types of stichocyte to be distinguished. By applying this stain on infected muscle sections after immunocytochemistry using (a) anti-PC BH8 monoclonal antibodies, (b) serum from mice that had been infected twice in the presence of 0.05% thiabendazole (to prevent reproduction by adult females) and then bled on day 7 post-reinfection, (c) serum from infected mice that were bled on day 14 postinfection, or (d) serum from infected mice that were bled on day 42 postinfection, we found (1) that PC is an abundant structural epitope on the hypodermis/muscle, genital primordium and intestinal tract but is absent from the cuticle and stichosome; (2) that the principle secretory cells of adult worms are delta- and beta-stichocytes, whereas those of migrating and encysted L1 larvae are alpha-stichocytes; and (3) that Trichinella antigens recognized in the encysted phase of the parasite's life cycle are present in parasitized myofibres in the sarcoplasmic matrix and in the nucleoplasm of hypertrophic nuclei. The significance of these findings is discussed.

Animals↗
PubMed1991

Specific immunosuppression by Trichinella: fine specificity and effect on lymphocyte function in vivo.

Muscle-phase Trichinella larvae depress the immune response of mice to the phosphorylcholine (PC)-bearing Trichinella antigen FCp without affecting responses to other PC-bearing or non-PC antigens. The depressive activity is independent of antigen dose and Trichinella species and, in adoptive cell transfer experiments with lethally irradiated recipient mice, depended on the state of the recipient (infected recipients had a depressed response even a month after their encysted larvae had been killed and regardless of whether the donor had been exposed to FCp) but not on the state of the transferred cells. We conclude that lymphocytes are not permanently altered by the depressive action, that the agent responsible persists in the host at least a month after the death of the encysted Trichinella larvae, and that the alteration does not eliminate lymphocyte immunological memory.

Animals↗